The function of the human nervous system is dependent upon billions of nerve cells. A primary mechanism by which this vast number of cells communicates involves chemical synapses - specialized junctions where a small molecule neurotransmitter released by one cell binds to and activates receptors on an adjacent cell. In order for this cycle of neurotransmission to rapidly and faithfully repeat, the neurotransmitter must be cleared or removed from synapses. There are a large number of therapeutic drugs and a wide array of illicit compounds that modulate transporter function, including antidepressants, cocaine and amphetamines. At most chemical synapses, the removal of transmitter is accomplished by integral membrane proteins called transporters. In many cases, such as with glutamate, GABA, glycine and the biogenic amine transporters, the transporter proteins harness ion gradients established by ATP-dependent pumps to thermodynamically drive or pump transmitter into adjacent cells; these proteins are commonly referred to as ion-coupled symporters. In other cases, such as with the glutamate/cystine exchanger, the transporter protein obligatorily exchanges one substrate (glutamate) for another (cystine); these transporters are generally referred to as antiporters. Because both symporters and antiporters are highly hydrophobic integral membrane proteins, studies of their atomic structures by x-ray diffraction methods have proven difficult. I propose to carry out high resolution crystallographic studies of bacterial orthologs of neurotransmitter symporters and antiporters and, in combination with complimentary functional studies, develop molecular mechanisms for the function of these crucial transporter proteins. In addition, I propose to commence structural studies of eukaryotic neurotransmitter transporters facilitated by new technology developed in my laboratory. By accomplishing the proposed studies, we will not only learn how these proteins function, but we will also have the fundamental information for the development of new compounds to treat a wide range of neurological diseases and disorders. PUBLIC HEALTH RELEVANCE: Integral membrane transport proteins remove chemical messengers or neurotransmitters from special junctions between nerve cells called synapses and their dysfunction is associated with numerous neurological diseases and disorders. The transport proteins are the targets of both therapeutic agents, such as antidepressants, and illicit substances, such as cocaine. The aims of the work proposed in this application are to determine the molecular structure and function of these important transporter proteins.